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Deep three-photon imaging of the brain in intact adult zebrafish


Behaviors emerge from activity throughout the brain, but noninvasive optical access in adult vertebrate brains is limited. We show that three-photon (3P) imaging through the head of intact adult zebrafish allows structural and functional imaging at cellular resolution throughout the telencephalon and deep into the cerebellum and optic tectum. With 3P imaging, considerable portions of the brain become noninvasively accessible from embryo to sexually mature adult in a vertebrate model.

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Fig. 1: Imaging deep into zebrafish and Danionella brains.
Fig. 2: Functional imaging of main brain regions in intact adult zebrafish.

Data availability

The imaging and scattering length data sets that support the findings of this study are available from the corresponding author upon reasonable request.

Code availability

The software codes used for the analyses are available from the corresponding author upon reasonable request.


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The work was supported by grant nos. NSF DBI-1707312, NIH 1U01NS103516, NSF IOS-1457108 and NIH S10OD012287. We thank C. Walcott (Cornell University) for photographic assistance, D. Mclean (Northwestern University) for his drawing skill, M. Farrar (Messiah College) for the mTdTomato construct and T. Porri (Cornell University) for performing the CT scans.

Author information




D.S., K.E.K., D.M.C, D.G.O., N.A., C.X. and J.R.F. contributed to experimental design and conceptual ideas. D.S and D.G.O. built and maintained the three-photon microscopes. K.E.K, D.M.C., D.S., D.G.O. and N.A. performed three-photon imaging experiments of zebrafish. Analysis was conducted by D.M.C., D.S., N.A. and K.E.K. R.T. and N.A. performed Danionella three-photon imaging. A.B. and J.R.F. supervised Danionella imaging and analysis. J.R.F. and D.M.C. prepared the manuscript with assistance from K.E.K. and input from all authors. J.R.F. and C.X. supervised all aspects of the project.

Corresponding authors

Correspondence to Chris Xu or Joseph R. Fetcho.

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Competing interests

The authors declare no competing interests.

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Peer review information Nina Vogt was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Structural features visible in optical sections with THG that facilitate navigation through the brain.

A. Telencephalic region, showing from left to right: the skin surface features; the transition into the brain as shadows of neurons become visible; and deeper in the telencephalon where the shadows of the neurons clustered at the midline are evident, as are scattered neurons throughout the section. B. Optic Tectum, showing from left to right: axon bundles on the surface of the tectum just below the skin and skull, with scales on the skin visible; deeper axon bundles in fiber layers of the tectum; densely packed shadows of cell bodies deep in the tectum; a major blood vessel lying adjacent to the ventricle deep in tectum. OT N= 16, Tel N=12.

Extended Data Fig. 2 Third harmonic generation images at different depths in the cerebellar and optic tectal areas.

Squares on brain diagrams on the right show approximate areas of imaging, and dashed lines represent approximate brain midlines. a, Cerebellar region from shallow to deep imaging. At 60 μm, some skin is visible (red asterisk) due to the curvature of the brain, as well as several blood vessels (red triangles) under the skin and above the skull. At 120 μm, skull features are evident. Deeper into the brain, fiber tracts predominate (red arrows). b, Optic tectal region from shallow to deep. At 147 μm, some skull is evident (green square) as well as some brain. Tectal fiber tracts are evident (green asterisk) at multiple depths (see 147, 245 and 378 μm) with a thick commissural fiber tract present (green triangle) at 490 μm. At deeper layers fiber tracts predominate (green arrows). CB N=10, OT N=16.

Extended Data Fig. 3 Signal to Background ratio.

Signal-to-background ratio (SBR) in shallow and deep regions in three areas of the brain. ac, Fluorescence intensity profiles along the lines across GCaMP6s-labeled neuronal nuclei (N=1 each). df, Fluorescence intensity profiles along the lines across blood vessels labelled with fluorescein (N=2 each). To produce each plot, a five-pixel-wide line was drawn across the blood vessel or nucleus. The 5 pixel values were averaged to get an intensity value for the corresponding position along the line. The x axis corresponds to the lines shown on the images to the right. Scale bar 50 μm.

Extended Data Fig. 4 3P Axial resolution measurement.

a, 3P image of a small fluorescein-labeled blood vessel (blue arrowhead) imaged about 200 μm deep into the optic tectum. Image stack was taken with 0.3 μm axial steps at 3 µs pixel time (512x512 pixels/frame) and 4 times averaging. b, Intensity profile along the axial direction fitted with a Gaussian curve to characterize the upper bound of axial resolution. The full width at half maximum of the Gaussian fit is 2.9 μm. N=1.

Extended Data Fig. 5 Example photon counts from telencephalic neurons.

Rows ac, show neurons at different depths. In each panel, the plots in magenta and green show photon counts from two neurons at that depth, with the monitored neurons marked with m and g in the optical section shown at the right. Telencephalon functional imaging N= 9.

Extended Data Fig. 6 Raster plot of photon counts for all of the neurons shown in main Fig. 2.

Neurons were monitored at different depths in: A. telencephalon, B. optic tectum and C. cerebellum. Functional imaging: Tel N = 9, OT N = 6, CB N = 2.

Extended Data Fig. 7 Experimental setup.

(a) schematic drawing of the excitation sources and the optical path of the imaging setup. OPA – optical parametric oscillator, HWP – Half wave plate, PBS – Polarizing Beam splitter, DM – Dichroic mirror, PMT – Photomultiplier tube. inset(i) - Optical path for the prism compressor for the 1300 nm setup. inset(ii) - Optical path for the silicon plate compressor for the 1700 nm setup.

Supplementary information

Supplementary Information

Supplementary Table 1

Reporting Summary

Video 1

Blood flow imaged with THG at 8.5 Hz.

Video 2

Video of nonlinear damage induced by excessive laser intensity.

Video 3

Video from a montage of z stacks through the depth of telencephalon on one entire side of the brain from superficial to deep.

Video 4

Three-dimensional video of one half of the telencephalon made from the z stacks shown in Supplementary Video 3.

Video 5

Functional imaging with GCaMP6s 300 µm deep into the optic tectum for approximately 6 min in a region with densely packed neurons from the same optical section and data shown in Fig. 2b.

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Chow, D.M., Sinefeld, D., Kolkman, K.E. et al. Deep three-photon imaging of the brain in intact adult zebrafish. Nat Methods 17, 605–608 (2020).

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